Luca Rovatti

Protein alkylation in the presence/absence of thiourea in proteome analysis: a matrix assisted laser desorption/ionization-time of flight-mass spectrometry investigation.

Although it is highly recommended that reduction and alkylation of free –SH groups in proteins should be performed prior to any electrophoretic step (including the first isoelectric focusing/immobilized pH gradient (IEF/IPG) dimension), it is here reported that one component of the sample solubilization cocktail adopted recently (namely thiourea) strongly quenches such alkylation process (as typically carried out with iodoacetamide, IAA).The present matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) analysis demonstrates that thiourea is an effective scavenger of IAA, since its sulfur atom reacts as efficiently as the ionized, free –SH group of Cys in proteins at alkaline pH values (pH 8.5–9.0). As a result of this reaction, free IAA is quickly depleted by thiourea, via the formation of an intermediate adduct, which is rapidly deamidated to form the cyclic compound thiazolinidone monoimine. This reaction strongly competes with the direct addition reaction of IAA onto the –SH group in proteins, resulting in poorly alkylated proteins. It is, therefore, recommended that, whenever possible and compatible with the type of sample, thiourea should be omitted from the solubilizing cocktail in proteome analysis. However, after proper sample reduction and alkylation, thiourea can be incorporated into the IEF/IPG gel, where it will have the beneficial effect of augmenting protein solubility at their pI values and scavenging the excess of free IAA.

Structural and conformational variants of human β2-microglobulin characterized by capillary electrophoresis and complementary separation methods

The small (Mr = 11729) serum protein beta2-microglobulin is prone to precipitate as amyloid in a protein conformational disorder (PCD) that occurs in a significant number of patients on chronic hemodialysis. Analyses by capillary electrophoresis (CE) were undertaken to study beta2-microglobulin conformations under native separation conditions and showed an apparent heterogeneity of purified preparations when the sample matrix included organic solvents such as acetonitrile, trifluoroethanol and ethanol. We here present LC-MS, CE-MS, and CE studies of changes of separation profiles as a function of capillary temperature, organic solvent concentration, and analysis time. The results suggest that the apparent beta2-microglobulin heterogeneity observed by CE is caused by two distinct protein conformations that are present in beta2-microglobulin under partly denaturing conditions and that Met99-oxidized and normal (i.e. nonoxidized) beta2-microglobulin behave similarly with respect to the potential to attain this alternative conformation. CE is an attractive method to study early and intermediate soluble folding variants that may be involved in PCDs and CE thus may have an important role as a tool for understanding other PCDs characterized by amyloid deposition.

PEPTIDE NITRATION BY PEROXYNITRITE : CHARACTERISATION OF THE NITRATION SITES BY LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY

A number of recent studies have demonstrated that peroxynitrite (ONOO−) can react with a host of biomolecules, particularly those containing aromatic amino acid residues, resulting in a number of modifications which have been found in association with diverse pathological conditions. Electrospray ionisation tandem mass spectrometry with and without liquid chromatographic separation has been used to examine a series of model peptides following their treatment with peroxynitrite at physiological pH. The mass spectra of sequences containing two tyrosine residues showed the formation of both mononitrated and dinitrated species, while those with phenylalanine showed no detectable nitration. Tryptic digests of two of the investigated peptides were also examined by liquid chromatography/electrospray mass spectrometry, which yielded further information on the competition for NO2 by multiple nitration sites within a given sequence. In addition, tryptic digests of the mononitrated component of sequences containing tyrosine and tryptophan indicated that mononitration was limited to tyrosine. Furthermore, some of the presented data indicate that, as well as nitrotyrosine and nitrotryptophan formation, the reaction of ONOO− can result in oxidation as well as the formation of labile adducts with NO2. Copyright

A multi-technique approach using LC-NMR, LC-MS, semi-preparative HPLC, HR-NMR and HR-MS for the isolation and characterization of low-level unknown impurities in GW876008, a novel corticotropin-release factor 1 antagonist.

A multi-technique approach was applied in order to fully characterize four low-level unknown impurities of GW876008, a novel CRF(1) receptor antagonist. Liquid chromatography (LC)-NMR spectroscopy was used in combination with LC-MS to obtain detailed information regarding the structure of the two major impurities present in batches of GW876008 and observed in the first synthetic scale-up for preclinical use. Two additional impurities were unexpectedly found at greater levels in a large scale synthesis for clinical use and their structure was elucidated by means of high resolution (HR)-MS and HR-NMR, after a small scale preparative HPLC purification step. This structural information was useful in terms of shedding light on the typical impurity profile of this new chemical entity with the aim to support the early development package for Phase I clinical studies.

Liquid chromatography/tandem mass spectrometry to monitor acrylamide adducts with bovine β‐lactoglobulin B

Complexation of acrylamide with bovine β-lactoglobulin B and some of its tryptic fragments have been examined by liquid chromatography coupled to tandem mass spectrometry. Such complexation was investigated both in the presence and in the absence of dithiothreitol as a reducing agent. Under the latter conditions, the intact protein exhibited a single cysteine– acrylamide complex which both the present work and previous studies attribute to Cys160. The involvement of this particular residue is tentatively attributed to an intramolecular disulphide exchange which results in its disengagement from the S–S bridge to offer a free SH group for reaction with the acrylamide monomer. In the absence of dithiothreitol, both free and complexed cysteine-containing tryptic fragments were present, while in its presence, one of the tryptic fragments, which contains three cysteine residues was fully absent, instead a part of this fragment containing two cysteines complexed with two acrylamide monomers was observed. The absence of any analytical information in the literature regarding the latter complexes underlines the potential of liquid chromatography coupled to mass spectrometry in the characterization of this commonly occurring modification.

Investigation of potential degradation products of a newly synthesised β-lactam antibiotic by multi-stage liquid chromatography–electrospray mass spectrometry

Abstract This work describes the use of multi-stage liquid chromatography–electrospray mass spectrometry to characterise four potential degradation products associated with a suspension containing a newly synthesised β-lactam antibacterial compound. Initial identification of these degradates was obtained by analytical LC in which the mobile phase contained cetyltrimethylammonium bromide as ion-pairing agent and ammonium phosphate buffer, both of which are highly involatile making them incompatible with electrospray mass spectrometry. The use of multi-stage liquid chromatography maintained the initial LC conditions necessary for the desired separation and at the same time facilitated the use of mass spectrometry detection.

Crudes of synthesis of neuropeptide Y and β‐amyloid peptide examined by liquid chromatography/electrospray tandem mass spectrometry

The two peptides, neuropeptide Y (M(r) = 4254) and beta (1-39) amyloid (M(r) = 4230) are attracting pharmaceutical and biomedical interest for different yet equally important reasons. The first is recognized for its various physiological functions in the peripheral and central nervous system, while the second has been identified as one of the components of the cerebral amyloid deposits characteristic of Alzheimers disease. High level purity of both peptides is considered of prime importance for correct interpretation of data obtained by biological and pharmaceutical assays and binding tests. Solid-phase synthesis of both peptides resulted in crudes of reaction containing both the target peptides as well as a number of undesired side products. The use of liquid chromatography/electrospray-tandem mass spectrometry (LC/ESI-MS/MS) furnished reliable information on the target peptides and provided sequencing information on a number of side products. Furthermore, LC/MS/MS data of doubly and triply protonated sequences yielded a number of C-terminal fragment ions exhibiting the loss of NH3 considered to be an important process for the understanding of fragmentation mechanism(s) of multiply protonated peptides.

Investigation of β‐Amyloid Peptide by On‐line High‐performance Liquid Chromatography Coupled to Electrospray Ionization Mass Spectrometry

beta(1-39) amyloid peptide is one of the components of the cerebral amyloid deposits that are characteristic of Alzheimers disease. Solid-phase synthesis of this peptide resulted in a fairly complex crude product containing both the target peptide and a number of side products. High-performance liquid chromatography coupled to electrospray ionization mass spectrometry allowed rapid and reliable identification of both the desired peptide and most of the side products which were found to have relative molecular masses above and below that of the target peptide.

Investigation of bombolitin complexes with mercury by liquid chromatography/tandem mass spectrometry

Solid-phase synthesis of (Cys)bombolitin peptide resulted in a crude reaction mixture containing the target peptide as well as a number of unknown products. The use of on-line liquid chromatography coupled to electrospray mass spectrometry (LC/ES-MS) gave a strong indication that these side-products were mainly complexes between the target peptide and mercury which was used in the form of mercury (II) acetate during the synthetic procedure. On-line LC/electrospray tandem mass spectrometry (ES-MS/MS) furnished reliable information on the amino acid sequences of both the target peptide and of the complexes with mercury or with mercury plus the b-mercaptoethanol used to eliminate the mercury at the end of the synthesis.# 1998 John Wiley & Sons, Ltd.

On-line liquid chromatography/electrospray tandem mass spectrometry to investigate acrylamide adducts with cysteine residues: implications for polyacrylamide gel electrophoresis separations of proteins.

Adduction between acrylamide and cysteine residues is a post-translational modification associated with proteins separated by gel electrophoresis. In the present article, three model peptides containing 2-4 cysteine residues were reduced with dithiothreitol, incubated with acrylamide monomers and examined by on-line liquid chromatography coupled to electrospray tandem mass spectrometry. Each of the solutions examined in this work revealed the presence of four distinct components: the free peptide, two different peptide-acrylamide 1:1 adducts involving two cysteine residues at different positions within the same sequence, and the peptide-acrylamide 1:2 adducts. The use of liquid chromatography allowed the separation of components which differed only by the site of complexation of acrylamide, while the application of tandem mass spectrometry furnished reliable sequencing information permitting the identification of most cysteine residues involved in such complexation.